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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plasmodium falciparum traffics a large number of proteins to its host cell, the mature human erythrocyte. How exactly these proteins gain access to the red blood cell is poorly understood. Here we have investigated the effect of protein folding on the transport of model substrate proteins to the host cell. We find that proteins must pass into the erythrocyte cytoplasm in an unfolded state. Our data strongly support the presence of a protein-conducing channel in the parasitophorous vacoular membrane, and additionally imply an important role for molecular chaperones in keeping parasite proteins in a ‘translocation competent’ state prior to membrane passage.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plasmodium falciparum is the causative agent of malaria tropica, the most serious form of human malaria, with several hundred million clinical cases each year (World Health Organization, 2008). Almost a million fatalities annually, mostly African children under the age of 5, can be attributed to this parasite (World Health Organization, 2008). In addition to the human suffering caused by this disease, malaria is also an important factor limiting sustained economic growth of the countries afflicted (Gallup and Sachs, 2001). A large part of the pathology of infection with falciparum malaria can be attributed to parasite-induced alterations in the host cell, the mature human erythrocyte. Infected host cells exhibit unusual adhesive properties, referred to as cytoadherence, allowing them to adhere to endothelial cells in the human host (Miller et al., 2002). The molecules responsible for this phenomenon are, in part, parasite-encoded proteins that are inserted into, or bind beneath the plasma membrane of the infected erythrocyte (Kilejian, 1979; Baruch et al., 1995; Smith et al., 1995; Su et al., 1995; Crabb et al., 1997; Wickham et al., 2001; Maier et al., 2008). How exactly these proteins gain access to the red blood cell is poorly understood.

Studies on protein transport to the host cell suggest that the early secretory pathway of P. falciparum is similar to that of higher eukaryotes, with proteins entering the endoplasmic reticulum (ER), based on presence of a functional N-terminal ER targeting signal (Benting et al., 1994; Wickham et al., 2001; Adisa et al., 2003). Such proteins are then thought to traffic along the secretory pathway in vesicles that eventually fuse with the parasite plasma membrane, releasing their cargo into ‘extracellular space’. While, in other systems, protein release at the plasma membrane is the last step in the secretory pathway, the situation in the P. falciparum-infected erythrocyte is different. Within the host cell the parasite resides within a membrane-bound compartment, referred to as the parasitophorous vacuole (PV) (Lingelbach and Joiner, 1998). Secreted proteins released at the parasite plasma membrane, but destined for a location within the host cell thus find themselves ‘trapped’ in the lumen of the PV, and must therefore be translocated across the membrane of the parasitophorous vacuole (PVM). This sequential transport pathway has been referred to as the ‘two-step’ model, and seems to be governed by a conserved pentameric transport sequence referred to as the Plasmodium export element (PEXEL) or host targeting signal (HT) (Ansorge et al., 1996; Baumeister et al., 2001; Hiller et al., 2004; Marti et al., 2004). The mechanistics of this membrane translocation process remain poorly understood, although models have been proposed to account for this unusual protein transport step, several of which support the existence of a membrane-bound protein-conducting channel (PCC) within the PVM. To better understand how parasite proteins translocate to the host cell, we have applied a previously described system based on the murine dihydrofolate reductase (mDHFR) to analyse whether transport across the PVM is dependent on the folding state of proteins (Eilers and Schatz, 1986). We find that transport of reporter constructs containing the mDHFR was, upon addition of DHFR-stabilizing agents, blocked within the lumen of the PV. This transport block was dose dependent and reversible. Our findings strongly suggest that parasite proteins destined for transport to the host red blood cell must translocate across the PVM in an unfolded configuration. These data are suggestive of a rate-limiting PCC-mediated step in the transport of parasite-encoded proteins to the erythrocyte cytosol. The data are discussed in regard to the nature of the putative vacuolar protein-conducting channel (VPCC), and the requirements for substrate proteins.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Expression of a mDHFR–green fluorescent protein (GFP) reporter in blood-stage parasites

We initially transfected blood-stage P. falciparum with expression constructs encoding a mDHFR–green fluorescent protein (GFP) reporter construct (Fig. 1A). A drug-resistant parasite population was established after 23 days. Fluorescence microscopy of this transfectant line (referred to as 3D7DG) verified that the GFP moiety of the fusion protein was able to efficiently form a fluorescent chromophore (Fig. 1B). As expected for a reporter protein lacking any targeting information, the GFP signal could be detected only within the parasite. Western blot analysis using anti-GFP antibodies verified that 3D7DG parasites express a protein of the expected molecular size (46.5 kDa, Fig. 1C).

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Figure 1. Expression of a DHFR–GFP reporter in P. falciparum. A. Structure of the DHFR–GFP reporter protein. Blue, DHFR; green, GFP. B. Epifluorescence microscopy of erythrocytes infected with 3D7DG. BF, bright field. C. Western blot of proteins extracts derived from 3D7DG. A total of 1 × 107 cells were loaded and probed with anti-GFP antibodies. Size marker in kDa.

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mDHFR can be stabilized in vitro by the addition of membrane permeant folate analogues, including WR99210

Stabilized, folded mDHFR is highly resistant to protease digestion, whereas unfolded mDHFR can be digested to completion with various proteases (Hausler et al., 1996; Backhaus et al., 2004). We wished to demonstrate that the mDHFR of our reporter protein can be stabilized by folate analogues, and carried out a protease protection assay. We prepared cytosolic protein extracts from transfectant line 3D7DG which were incubated in the presence or absence of the membrane permeant folate analogues aminopterin (AP) or WR99210 (WR). Initial experiments showed it unnecessary to add exogenous proteases to the assay, as endogenous parasite proteases also resulted in a high level of proteolytic degradation (Fig. S1A and B). As a negative control, we used dimethyl sulphoxide (DMSO). Following incubation for 14 h at 37°C, we could detect a characteristic size shift on Western blots (Fig. 2). In samples incubated in the absence of either AP or WR, the initial 46.5 kDa protein could no longer be detected, but in its place a c. 27 kDa band appeared, corresponding to GFP alone, which is highly resistant to protease digestion (Chiang et al., 2001) (Fig. 2). Addition of either AP or WR to the digestion reaction resulted in strong inhibition of proteolytic processing of the mDHFR–GFP fusion protein (Fig. 2), as did inclusion of protease inhibitors [protease inhibitor cocktail (PIC)]. Addition of DMSO had no effect on either stability of the fusion protein or protease activity, as evidenced by the complete degradation of P. falciparum heat shock protein 70 (PfHSP70) (Fig. 2). Western blot analysis using anti-mDHFR antibodies verified that the size shift upon proteolytic degradation is not due to cleavage between the mDHFR and GFP parts of the reporter protein, but due to total degradation of the mDHFR (Fig. S1).

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Figure 2. Protease protection assay. Protein extracts derived from 3D7DG were incubated at 37°C in the presence (+) or absence (−) of aminopterin (AP), WR99210 (WR), DMSO and/or protease inhibitor cocktail (PIC). A total of 1 × 107 parasite cell equivalents were then analysed by Western blot using anti-GFP and anti-PfHSP70 antibodies. t = 0 refers to time point 0, t = 14 to a 14 h incubation. Size markers in kDa.

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These results demonstrate that mDHFR–GFP is susceptible to protease treatment, but that this sensitivity can be largely reversed by addition of AP or WR, resulting in stabilization of the mDHFR moiety of the fusion protein.

Based on these results, for all further experiments we used WR as the stabilizing agent.

Transport of a mDHFR-containing chimeric reporter protein to the host erythrocyte is blocked by protein stabilization

We have previously demonstrated that a chimeric reporter protein consisting of the first 80 amino acids of the STEVOR protein fused to GFP is transported to the cytoplasm of the host erythrocyte (Przyborski et al., 2005). We generated a transfectant parasite line expressing a fusion protein consisting of the first 80 amino acids of STEVOR fused to mDHFR and GFP (Fig. 3A, referred to as 3D7SDG). Western blotting using anti-GFP antibodies confirmed expression of the reporter protein at the correct molecular mass [51 kDa, taking account of cleavage of the PEXEL pre-sequence (Fig. 3B)]. Importantly these parasites are resistant to the antiparasitic effects of WR, as they also express a mutant human DHFR from the episomally maintained plasmid. Parasites were then grown in the absence or presence of 5 nM WR and subjected to analysis by fluorescence microscopy. In the presence of WR (thus stabilizing the mDHFR protein), a fluorescent signal could be detected in a ‘ring’ structure around the body of the parasite (Fig. 3C, top), indicative of a localization within the lumen of the PV. Upon allowing the parasites to pass through one developmental cycle (48 h) without WR, the fluorescent signal changed dramatically. Now, the majority of the fluorescent signal was seen within the erythrocyte cytoplasm (Fig. 3C, bottom), with a small amount of fluorescence detected surrounding the parasite and within the food vacuole. This food vacuolar fluorescence results from non-specific uptake of GFP chimera from the red blood cell (Wickham et al., 2001; Przyborski et al., 2005).

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Figure 3. Expression of a STEVOR–DHFR–GFP reporter. A. Structure of the reporter. Red, STEVOR N-terminus (amino acids 1–80); blue, DHFR; green, GFP. B. Western blot of protein extracts derived from 3D7SDG. A total of 1 × 107 cell equivalents were loaded and probed with anti-GFP antibodies. Size marker in kDa. C. Epifluorescence microscopy of erythrocytes infected with 3D7SDG. Parasites were cultivated in either the presence (+WR) or absence (−WR) of 5 nM WR99210 and subjected to live-cell imaging. D. Streptolysin O lysis of 3D7SDG-infected cells. Following lysis, infected cells were separated into a pellet (P) and supernatant (S) fraction and subjected to Western blot analysis using anti-SERP and anti-GFP antibodies. Size markers in kDa. The c. 25 kDa smear in the supernatant fraction is due to unspecific reaction of the chemoluminescent substrate with haemoglobin.

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To confirm our microscopical observations, we carried out cell subfractionation experiments using the bacterial pore-forming protein streptolysin O (SLO), which only lyses the erythrocyte plasma membrane, while leaving the PVM intact (Ansorge et al., 1996). Following SLO treatment, infected erythrocytes were separated into a supernatant (soluble erythrocyte proteins) and pellet fraction (containing proteins from the PV and parasite cytoplasm) and identical cell equivalents analysed by Western blotting using anti-GFP antibodies. This experiment reveals that, in infected erythrocytes grown in the presence of WR, all of the reporter protein can be detected in the SLO pellet fraction (Fig. 3D, left). A further, low-intensity < 50 kDa GFP band could also be detected in this fraction, possibly the result of low-level proteolysis in the PV. In stark contrast, in infected cells cultivated without WR, a large proportion of the chimeric protein is found in the SLO supernatant fraction (Fig. 3D, right). Both SLO pellet fractions also revealed a previously reported < 30 kDa GFP degradation product (Przyborski et al., 2005). The PV marker protein SERP (serine-rich protein) can be detected only in the SLO pellet fraction, showing that the PV remained intact during the fractionation procedure (Fig. 3D). Importantly, the reporter protein was detected at the same size in both samples derived from parasites grown in the presence or absence of WR, showing that possible cleavage and processing of the c. 50-amino-acid N-terminal signal peptide and PEXEL pre-sequence in the ER is the same in both samples, and is not influenced by folding of the mDHFR.

Folding of the mDHFR might cause aggregation of the fusion protein, thus blocking transport. To control for this, we carried out ultracentrifugation on parasite extracts derived from cells cultured in the presence or absence of WR. Identical numbers of cells were analysed by Western blotting with anti-GFP antibodies. We could find no evidence that mDHFR folding compromised solubility of the reporter protein (Fig. S2).

Transport of a glycophorin-binding protein 130 (GBP130) mDHFR–GFP fusion protein is also blocked by stabilization of the mDHFR

The 3D7SDG parasite line expressed a reporter containing N-terminal targeting information derived from STEVOR, a membrane-bound protein (Cheng et al., 1998; Kaviratne et al., 2002; Przyborski et al., 2005). As the possibility exists that soluble proteins may be trafficked through a different pathway, we then studied the transport of another fusion protein, this time containing the N-terminal targeting region derived from a soluble protein. For this purpose we chose to use the first 150 N-terminal amino acids of the glycophorin-binding protein 130 (GBP130), fused to mDHFR and GFP (Fig. 4A). The first 99 amino acids of GBP contain a recessed hydrophobic signal sequence, and have previously been shown to drive trafficking of reporter constructs to the host erythrocyte (Marti et al., 2004). After transfection and selection, resistant parasites appeared after 28 days. Once again, we cultivated these parasites (referred to as 3D7GDG) in the presence or absence of WR. In infected erythrocytes grown under WR pressure, the GFP fluorescence was limited to a structure resembling the PV (Fig. 4B, top). Removal of WR from the culture medium for a period of 48 h resulted in transport of the GFP reporter to the cytoplasma of the host erythrocyte (Fig. 4B, bottom). Colocalization experiments using the PV marker protein PfPV1 verified the GFP-labelled structure in WR+ parasites as the lumen of the PV (Fig. 4C). Furthermore, SLO fractionation of infected erythrocytes revealed that, in cells grown under WR pressure, all of the GFP signal could be detected in the SLO pellet fraction (Fig. 4D). Removal of WR allowed transport of a large amount of the GFP chimera to the host cell cytoplasm, although a GFP signal could also be detected in the SLO pellet fraction (Fig. 4D). The significance of the vacuolar GFP fraction in these cells is discussed in later sections of this report. The integrity of the PVM was verified by testing all fractions with anti-SERP antisera. As expected, SERP could only be detected in the SLO pellet fraction (Fig. 4D). To verify complete host cell lysis, we analysed all fractions for the presence of human heat shock protein 70 (HsHSP70) which is present in large amounts in the mature erythrocyte (Banumathy et al., 2002; Pasini et al., 2006). HsHSP70 can be detected almost exclusively in the SLO supernatant fraction lysis (Fig. 4D, bottom). To control integrity of the parasite plasma membrane, we analysed the distribution of PfAldolase, a marker protein for the parasite cytoplasm (Fig. 4D). Aldolase is, as expected, found only in the SLO pellet fraction. The possibility existed that addition of WR blocked (through unspecific effects) the transport of PEXEL/HT-containing proteins to the host erythrocyte. For this reason, we also analysed fractions for the presence of endogenous GBP130. In cells cultured either in the presence or in the absence of WR, GBP130 could be detected in both the SLO supernatant and pellet fractions, consistent with previous reports (Ansorge et al., 1996), suggesting that the translocation arrest observed in these cells is due to a specific mDHFR-related effect.

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Figure 4. Expression of a GBP130–DHFR–GFP chimera. A. Structure of the reporter. Orange, GBP130 N-terminus (amino acids 1–150); blue, DHFR; green, GFP. B. Epifluorescence microscopy of erythrocytes infected with 3D7GDG. Parasites were cultivated in either the presence (+WR) or absence (−WR) of 5 nM WR99210 and subjected to live-cell imaging. The nucleus is stained with Hoechst 33258 (blue). C. Colocalization of the reporter protein with the PV marker PfPV1. Cells grown in the presence of 5 nM WR99210 were fixed under conditions that did not quench GFP fluorescence and subjected to indirect immunofluorescence labelling using anti-PfPV1 antibodies. The GFP signal (green) can be seen to substantially overlap with that of PfPV1 (red). The nucleus is stained with Hoechst 33258 (blue). D. Streptolysin O lysis of 3D7GDG-infected cells. Following lysis, infected cells were separated into a pellet (P) and supernatant (S) fraction and equal cell equivalents were subjected to Western blot analysis using anti-PfSERP, anti-GFP, anti-PfAldolase, anti-HsHSP70 and anti-PfGBP130 antibodies. Size markers in kDa.

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Taken together, these results convincingly show that stabilization of the mDHFR moiety blocks transport of reporter constructs across the PVM.

The transport arrest is dose dependent

Out initial experiments revealed that resistance to protease cleavage of the mDHFR/GFP moiety is dependent on the concentration of the stabilizing agent (data not shown). We hypothesized that if the transport block is truly due to stabilization of the mDHFR, it should be possible to control the transport arrest in a dose-dependent manner. Erythrocytes infected with 3D7GDG were grown in increasing concentrations of WR for 48 h prior to analysis via fluorescence microscopy. As previously shown, parasites grown in the absence of WR exhibited a strong fluorescent signal from the host erythrocyte (Fig. 5, 0 nM). This signal decreased in strength with increasing concentrations of WR, with a concomitant increase in the fluorescent signal seen within the PV (Fig. 5). Indeed the transport of the reporter construct appears to be extremely sensitive to WR concentration, as even a low concentration of 0.5 nM was sufficient to cause accumulation of a significant fraction of the chimeric protein within the PV (Fig. 5, middle). At concentrations > 2.5 nM, no signal could be detected from the host cell, with the entire fluorescent signal being seen in the PV (Fig. 5, bottom).

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Figure 5. Concentration dependence of the translocation arrest. Infected erythrocytes were cultivated in the presence of the indicated concentration of WR99210 for a period of 48 h. In the bottom panel, an erythrocyte is seen containing two parasites, only one of which appears to express the reporter protein. With an increasing concentration of WR99210, less reporter protein is transported to the erythrocyte cytoplasm, with an increase in the vacuolar fluorescence.

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Transport arrest is not due to overexpression of the reporter protein

As previously mentioned, our transfection vector pARL2 encodes a mutant human DHFR that confers resistance to WR, and this property is used to select for transgenic parasites (Crabb et al., 2004; Przyborski et al., 2005). For the purposes of our experiments, it was necessary to remove drug pressure from the parasite cultures for up to 48 h, allowing the parasites to complete one developmental cycle. In our hands, removal of drug pressure for two developmental cycles leads to a dramatic reduction in the expression of GFP reporter constructs (N. Gehde and J.M. Przyborski, unpubl. data), probably due to a reduced plasmid copy number and/or inefficient plasmid segregation. We wished to discount the possibility that drug pressure led to an overexpression of the reporter protein to the point that transport pathways are blocked and thus transport of the chimeric protein to the host erythrocyte is inhibited. To this end, we generated transgenic parasites expressing only the first 150 amino acids of GBP130, fused to GFP (Fig. 6A) (referred to as 3D7GG). Erythrocytes infected with 3D7GG cultivated in either the presence or absence of WR for a period of 48 h were examined by fluorescence microscopy. As can be seen in Fig. 6A, neither the presence nor absence of 5 nM WR in the culture medium led to an accumulation of the fluorescent reporter in the PV. In these cells, the fluorescent signal can be clearly detected in the cytoplasm of the infected erythrocyte (Fig. 6A, top and bottom). This result was also verified by SLO fractionation followed by Western blotting (Fig. 6B). In both samples, a large proportion of the reporter construct can be detected in the SLO supernatant (Fig. 6B, right side). Additionally, a fraction of the GFP fusion protein, including several GFP degradation products, can be detected associated with the SLO pellet fraction (Fig. 6B, left side). To verify that the PVM remained intact during the lysis procedure, we also performed Western analysis with antisera to the PV resident protein SERP. As expected, SERP could be detected almost exclusively in the SLO pellet fraction (Fig. 6B, top). These data suggest that protein expression levels alone cannot explain the transport arrest of reporter constructs and underscores the contribution of the mDHFR to the translocation arrest phenomenon observed above.

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Figure 6. Reporter protein overexpression does not lead to accumulation in the PV. A. Erythrocytes infected with 3D7GG were cultivated in the presence (+WR) or absence (−WR) of 5 nM WR99210 for a period of 48 h, then visualized by epifluorescence microscopy. In both cases, all of the fluorescent signal can be seen within the cytoplasm of the infected erythrocyte. B. SLO fractionation of cells in (A). Infected cells were subjected to SLO lysis, separated into a pellet (P) and supernatant fraction (S) and equal cell equivalents analysed by Western blotting with anti-GFP and anti-SERP antibodies.

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In vitro stabilization of the mDHFR is dynamic and reversible

We were interested in investigating the stability of mDHFR in complex with WR. We therefore isolated protein extracts (SLO pellet) from 3D7SDG grown under drug pressure, and incubated them for varying periods in the absence of WR. We omitted PIC from the reaction mixture, and followed the proteolytic degradation of the full-length reporter protein as an assay for mDHFR stabilization. As a control for the activity of the endogenous proteases, we monitored the proteolytic degradation of the vacuolar marker protein SERP (Fig. 7A, top). Following incubation of the samples for 1 h, the proportion of full-length fusion protein (51 kDa) can be seen to have significantly decreased compared with the control (compare t = 0, bottom with t = 1, bottom). As in the previous experiment, we could also detect a weak lower-molecular-mass band that reacted with anti-GFP antibodies, in addition to a < 30 kDa GFP band. Degradation of the full-length reporter protein is even more pronounced after a 4 h incubation (bottom, t = 4), with only a very small amount of either full-length or > 50 kDa truncated products detected. SERP was also rapidly degraded under these conditions (Fig. 7A, top). Thus, the mDHFR–WR complex rapidly disassociates, allowing unfolding of the mDHFR and its proteolytic degradation.

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Figure 7. Stability of the mDHFR–WR complex. A. Protein extracts derived from 3D7SDG parasites grown in the presence of WR99210 were incubated at 37°C without WR99210 for the time (hours) indicated. Following this, equal cell equivalents were analysed by Western blotting with anti-GFP and anti-SERP antibodies. B. Infected erythrocytes containing 3D7GDG parasites grown either with (+) or without (−) WR pressure were removed from culture, washed and returned to culture with or without WR. Parasites were allowed to grow for 6 h, and were then visualized by live-cell microscopy. −/− and +/+ are shown for reference.

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Previous earlier reports have suggested that parasite-encoded proteins destined for transport to the cytoplasm of the host erythrocyte are first secreted into the lumen of the PV, from where they then translocate across the PVM (Ansorge et al., 1996; Wickham et al., 2001; Hiller et al., 2004; Marti et al., 2004). With this in mind, we were interested to see whether it was possible to ‘chase’ GFP reporters arrested in the PV across the PVM by removal of WR. In addition, we also were interested to investigate how quickly WR could produce a measurable effect on transport of the GFP chimera to the host cell. Erythrocytes infected with 3D7GDG cells cultivated in either the presence or absence of WR were removed from culture, washed in medium without WR, split into two aliquots and then returned to culture for a period of 6 h, again in the presence or absence of drug pressure. These data are presented in Fig. 7B. In infected erythrocytes previously cultivated in WR-containing media (Fig. 7B, referred to as +/−), culture without WR for a period of 6 h resulted in an obvious change in the fluorescence pattern. Although a strong fluorescent signal can still be detected within the PV, a significant portion of the GFP signal can also be visualized within the cytoplasm of the host erythrocyte (Fig. 7B, referred to as +/−). Conversely, addition of WR pressure to parasites previously cultured without WR (Fig. 7B, referred to as −/+) led to a distinct GFP fluorescence within the lumen of the PV. Therefore, passage through an entire developmental cycle is not necessary to reverse the effect of WR.

Only newly synthesized reporter protein can pass through the vacuole

Our initial experiments revealed that (i) removal of WR allows protein transport across the PVM and (ii) the DHFR–WR complex rapidly disassociates. However, recovery of protein transport across the PVM was not directly evident upon removal of WR, but required several hours to be detectable. One explanation for this observation is that only newly synthesized GFP chimera can be translocated across the PVM. To investigate this, we initially attempted to repeat our ‘chase’ experiments in the presence of the protein synthesis inhibitor cyclohexemide. Unfortunately, addition of cyclohexemide [which kills the parasite within a very short time (Baumeister et al., 2001)] to parasite cultures for the period of the assay (6 h) led to parasites with abnormal morphology (N. Gehde and J.M. Przyborski, unpubl. obs.). As an alternative, we decided instead to use the fungal metabolite Brefeldin A (BFA), which blocks protein secretion via the ER/Golgi system, and has been extensively used to study protein transport in the P. falciparum system (Misumi et al., 1986; Oda et al., 1987; Elmendorf and Haldar, 1993; Benting et al., 1994; Wiser et al., 1999; Wickham et al., 2001). 3D7GDG parasites grown in the presence of WR were washed in WR-free media, re-suspended in complete media containing 5 μg ml−1 BFA and returned to culture for a further 6 h. Following this, infected cells were examined by fluorescence microscopy. In contrast to untreated cells (Fig. 8, top), infected erythrocytes incubated in either the presence or absence of WR, and presence of BFA showed fluorescence only in the PV, with no fluorescence signal detectable within the cytoplasm of the infected host cell (Fig. 8, middle and bottom). In addition, a fluorescent signal was evident within the parasite, in close apposition to the nuclear stain, probably representing the ER (Fig. 8, bottom, white arrow). These data, taken together with the presented data on the dynamics of mDHFR stabilization, provide evidence that only newly synthesized reporter is capable of being translocated across the PVM into the host cell.

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Figure 8. Effect of BFA on protein transport. Infected erythrocytes containing 3D7GDG parasites grown in the presence of WR were removed from culture, washed and then returned to culture with the addition of either 5 nM WR, 5 μg ml−1 BFA or both simultaneously. Parasites were allowed to grow for a further 6 h before imaging. The white arrow highlights a fluorescent perinuclear compartment, probably representing the parasites ER.

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The PV of 3D7GDG contains significant amounts of non-fluorescent GFP

In the absence of WR we could detect GFP in both the SLO supernatant and SLO pellet fractions derived from 3D7GDG-infected erythrocytes (shown in Fig. 4D). However, microscopic analysis of these cells did not reveal a GFP signal within the PV (Fig. 4B). The possibility existed that our failure to detect a GFP signal within the vacuole was due to the limits of light microscopy that could not resolve this structure due to the strong fluorescent signal emitted from the erythrocyte cytoplasm. To exclude this, we carried out SLO lysis under conditions that would allow us to directly visualize whether residual GFP fluorescence could be detected in the PV. As a control for these experiments we first carried out the analysis on cells grown in the presence of WR [which exhibited a strong fluorescent signal within the PV (Fig. 4B)]. Upon lysis of the host cell (Fig. 9A, 4.4 s) the fluorescent signal could still be visualized within the PV, and remained stable for the course of the experiment. In contrast, infected erythrocytes grown in the absence of WR exhibited initially a strong fluorescence within the host erythrocyte (Fig. 9B, 0.0 s). Upon lysis of the erythrocyte plasma membrane with SLO, this fluorescent signal diffused rapidly into the surrounding media (Fig. 9B, 2.2 and 4.4 s; Movie S1). In these cells, no residual GFP signal can be detected coming from the PV (Fig. 9B).

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Figure 9. Live-cell imaging of SLO lysis. A. Infected erythrocytes containing 3D7GDG parasites grown in the presence of WR were allowed to settle onto the bottom of an imaging chamber. SLO (2 haemolytic units) was added, and cellular lysis visualized in both the GFP and differential interference contrast (DIC) channels. Note that even after lysis the PV fluorescent signal can be clearly seen. Time is indicated in seconds. B. Infected erythrocytes containing 3D7GDG parasites grown in the absence of WR were allowed to settle onto the bottom of an imaging chamber. SLO (2 haemolytic units) was added, and cellular lysis visualized in both the GFP and DIC channels. In this experiment, cell lysis was rapid, and the fluorescent signal within the infected erythrocyte dispersed rapidly following lysis. The panels on the right hand side have been false coloured to more clearly show the diffusion of the GFP signal around the erythrocyte. Time is indicated in seconds. A movie of this experiment is included as supporting information (Movie S1).

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Although GFP can only be detected in live-cell microscopy once the chromophore has properly formed, indirect immunofluorescence can be used to visualize GFP in fixed cells. We analysed the distribution of the GFP fluorescence signal using anti-GFP antibodies. In these cells, we were able to detect a fluorescent signal within the PV, as shown by the colocalization with the PV marker PfPV1 (Fig. 10, top). As an additional control, we carried out indirect immunofluorescence on fixed cells, having first removed the soluble protein content of the erythrocyte cytosol by SLO lysis. In these cells, no direct GFP fluorescence can be seen within the PV (Fig. 10, bottom, GFP); however, immune-labelling of GFP molecules with anti-GFP antibodies followed by a indocyanine 3 (Cy3)-coupled secondary antibody allows visualization of a non-fluorescent vacuolar GFP species (Fig. 10, bottom, anti-GFP).

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Figure 10. Visualization of non-fluorescent GFP. Intact (top), or SLO lysed infected erythrocytes (bottom) were fixed and subjected to IFA analysis with anti-GFP/anti-PV1 antisera followed by fluorescent-coupled antibodies. In both cases, a ‘ring’ fluorescent signal can be seen surrounding the parasite. In the bottom panel, note that GFP can only be seen upon treatment with anti-GFP antibodies.

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Taken together, these data show that the vacuolar portion of GFP in these cells, although present, does not fluoresce under live-cell imaging conditions, most likely due to the lack of an active chromophore.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plasmodium falciparum is predicted to export over 250 proteins to the cytoplasm of the host erythrocyte (Hiller et al., 2004; Marti et al., 2004; 2005; Sargeant et al., 2006). Characterization of some of these proteins has revealed that they are potentially involved in diverse processes within the infected cell, including cytoadherence, stabilization of the host cell cytoskeleton, protein traffic and nutrient acquisition (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995; Crabb et al., 1997; Silva et al., 2005; Baumeister et al., 2006; Cooke et al., 2006; Maier et al., 2007; 2008; Pei et al., 2007). Earlier work demonstrated that soluble parasite proteins destined for the host erythrocyte pass transiently through the lumen of the PV before being secreted into the red blood cell (Ansorge et al., 1996; Baumeister et al., 2001). Later studies elucidated that this transport step (at least for many proteins) is mediated by a short conserved pentapeptide sequence found towards the N-terminus of proteins, referred to as PEXEL, HT or HCT (Hiller et al., 2004; Marti et al., 2004; Przyborski et al., 2005). Taken together, these studies allowed development of a working model of protein transport across the PVM, the basis of which relies on the presence of a membrane-bound VPCC within the PVM (Charpian and Przyborski, 2008). However, apart from theoretical considerations, no thorough mechanistic analysis has yet described the properties or indeed provided direct evidence for the existence of this protein translocon.

Here we have addressed this issue by studying how the folding state of proteins influences their translocation to the cytoplasm of the infected erythrocyte. The mDHFR can be stabilized in vitro by the addition of folate analogue such as AP. Expression of mDHFR fused to targeting signals for cellular subcompartments has previously been used to study protein transport and the protein translocons involved in the import of proteins into (among others) mitochondria, chloroplasts, glycosomes and peroxisomes (Eilers and Schatz, 1986; America et al., 1994; Hausler et al., 1996; Soll and Schleiff, 2004). Furthermore, the system has also been applied to study unconventional secretion of FGF-2 as well as ERAD-based protein retrotranslocation from the ER (Backhaus et al., 2004; Bhamidipati et al., 2005). These studies have revealed that PCCs, on the basis of their ability to deal with differently folded substrates, fall into several different classes. For example, while import of soluble proteins into the nucleus and peroxisomes seems to be unaffected by folding status of the target protein, import into mitochondria requires proteins to enter into a so-called ‘translocation competent’ state before passage across the membrane can occur (Neupert and Brunner, 2002; Platta and Erdmann, 2007; Stewart, 2007). As an unusual special case, the bacterial twin-arginine translocation pathway appears to require proteins to fold into their final tertiary or even quaternary structure before protein translocation takes place, and uses this criterion as a ‘quality control’ for correct protein folding and subunit assembly prior to protein export (Saier, 2006). Thus, the study of how folding affects protein transport can be used to identify which class of protein translocon is responsible for a particular translocation process.

We have previously suggested that PEXEL/HT exerts its influence at an earlier point in the secretory pathway than originally proposed (Lingelbach and Przyborski, 2006). During the preparation of this article, Chang et al. (2008) provided solid experimental evidence that PEXEL/HT acts as a recognition site for an ER resident endoprotease, which cleaves the N-terminal pre-PEXEL/HT sequence. The ‘new’ N-terminus of the protein is then acetylated. Although these data require us to re-think how exactly PEXEL/HT exerts its influence on protein transport, this current study is not concerned with the mechanistics of PEXEL/HT recognition, but rather the pathway taken by PEXEL/HT-containing proteins. Thus, for the purposes of this discussion, we shall still refer to ‘PEXEL/HT-containing proteins’ as a synonym for proteins whose transport is directed by this signal.

In this present study we have studied how transport of the model substrate protein mDHFR across the PVM is affected by induced folding of the protein into its stable tertiary structure. We find that stabilization of the mDHFR moiety results in a ‘translocation arrest’ within the lumen of the PV. To our knowledge, this is the first application of the mDHFR system to study transport processes in the P. falciparum-infected cell, and also the first time that the effect of protein folding on transport across the PVM has been addressed. Our results have important implications.

The original two-step model for transport of soluble parasite proteins to the host cell was based on the analysis of only one protein (GBP130), and the possibility could not be discounted that further proteins bypassed the PV and were translocated directly from the parasite to the host cell (referred to as the one-step model) (Ansorge et al., 1996). Our data strengthen the view that all soluble parasite proteins destined for the erythrocyte cytoplasm are initially secreted into the PV, from where they are then translocated across the PVM. This hypothesis is further supported by the fact that the one-step model relies on secretory vesicles containing parasite proteins fusing with ‘junctions’ at which parasite plasma and PVM meet. Although, based on our results, we cannot totally discount a role for vesicular transport, we suggest that protein folding is unlikely to have an effect on transport of soluble proteins carried by vesicular budding processes, but only on proteins that directly translocate across a biological membrane. Alternatively, it is possible that parallel protein-translocating pathways are present, differing in their requirements for protein unfolding.

A recent publication from Bhattacharjee et al. (2006) has suggested that PEXEL/HT acts as a recruitment signal for proteins to the Maurer's clefts, from where they then enter the host cell cytoplasm. Our present study, in contrast, suggests that parasite proteins translocated directly across the PVM, and are not required to first travel to the Maurer's clefts. This contradiction may be explained by the nature of the experiments performed. Thus, while Bhattacharjee and co-authors measured the steady state of protein localization, our experimental system was designed to measure dynamic transport processes, allowing us to follow the individual steps in protein transport from the parasite to the infected erythrocyte. Previous studies using fluorescence recovery after photobleaching (FRAP) have suggested that the lumen of the PV is not in full liquid continuity, and that subdomains of the PV exist which are not directly connected to each other (Adisa et al., 2003). Thus, an alternative interpretation of our data is that folding of the mDHFR protein hinders diffusion of the reporter protein between these PV subdomains. This would however seem unlikely, as the fluorescent pattern we observed in the PVM did not differ from that previously reported upon the targeting of a variety of GFP chimera to this compartment (Adisa et al., 2003; Hiller et al., 2004; Marti et al., 2004; Przyborski et al., 2005), suggesting that our mDHFR-containing reporter protein can freely diffuse into all subdomains of the PV lumen.

Our experimental system allows a dissection of the individual transport steps that soluble parasite proteins pass through before reaching their final localization. Based on analysis of parasites expressing mutant PEXEL/HT-containing reporter proteins, several authors have suggested that the lumen of the PV serves as a transitional compartment for protein transport to the host cell (Wickham et al., 2001; Hiller et al., 2004; Marti et al., 2004; Przyborski et al., 2005). Nevertheless, it could not be discounted that mutation of the PEXEL/HT signal could have led to proteins being directed into another transport pathway. One example of such aberrant targeting is exhibited by proteins targeted to the P. falciparum plastid. Dissection of targeting signals suggested that proteins destined for the apicoplast pass through the lumen of the PV (Cheresh et al., 2002). Other studies however revealed that, in fact, mutation or deletion of transit peptides caused proteins to follow the default secretory pathway, a route that is not followed by the endogenous (non-mutated) protein (Waller et al., 1998; 2000; Foth et al., 2003). By dissecting individual transport steps, our experiments show that GFP chimera containing canonical PEXEL/HT transport signals do in fact pass through the lumen of the PV, giving further support to a general two-step model for all soluble proteins targeted to the host erythrocyte, and strengthening the view that the lumen of the PV acts as an important protein-sorting compartment.

Earlier studies using GFP reporter constructs suggest that transport of proteins across the PVM is likely to be independent of protein folding, as GFP-containing reporter constructs can be translocated to the host cytoplasm (Wickham et al., 2001; Hiller et al., 2004; Marti et al., 2004). Upon folding, the GFP structure is that of a cylinder with a diameter of about 30 Å and a length of about 40 Å (Ormo et al., 1996; Yang et al., 1996). Given that the size of the pore region of the Sec61 ER translocon is estimated to be 15–20 Å (Hanein et al., 1996; Beckmann et al., 1997), that of the chloroplast TOC75 between 14 and 26 Å (Hinnah et al., 2002), and the mitochondrial TOM40 and TIM 20 Å (Kunkele et al., 1998; van der Laan et al., 2006), it is to be expected that fully folded GFP cannot be translocated through such channels. If we hypothesize that the VPCC is of a similar nature, then it is similarly unlikely that a full folded (and hence fluorescent) GFP molecule can pass through this channel. Our experimental data provide a possible solution to this mechanistic quandary. By studying the transport dynamics of protein transport following removal of the translocation arrest, we were able to show that only newly synthesized reporter proteins are capable of traversing the PVM. In addition, we could show that, in cells grown without WR, a significant amount of non-fluorescent GFP was present in the lumen of the PV. We believe that these observations can be explained by the folding dynamics of the GFP reporter. Formation of the GFP chromophore is a spontaneous process that requires no auxiliary protein factors and molecular oxygen. Although different GFP variants require different periods for chromophore formation, it can be said that GFP maturation is a relatively slow process that can take up to several hours. In cells grown without the presence of the mDHFR-stabilizing drug WR, newly synthesized reporter protein is rapidly transported along the default secretory pathway to the lumen of the PV, from where it is then quickly translocated into the host erythrocyte. In this situation, GFP molecules will only fully fold, reach maturity and form active chromophores once within the erythrocyte cytoplasm. In contrast, the addition of WR blocks transport of the reporter construct in the PV. In this case, GFP can mature, and thus fluoresces, also in the PV lumen. Upon removal of WR (and thus unfolding of the mDHFR moiety) and addition of BFA to block secretion of newly synthesized protein, we could not detect any translocation of this fluorescent vacuole-localized reporter protein pool into the host erythrocyte. Our data strongly suggest that our failure to ‘chase’ the reporter out of the vacuole is due to the presence of a fully folded GFP molecule within the chimera, which cannot pass through the VPCC. One important consequence of these data is that they reveal a novel mechanism for protein sorting in P. falciparum, based purely on the folding rate of substrate proteins, similar to that already described for the major adenylate kinase of mitochondria (Magdolen et al., 1992; Angermayr et al., 2001). This mechanism may, for example, be able to explain the dual localization of the PEXEL/HT-positive protein GBP130, which is found in high levels in both the PV and erythrocyte cytoplasm. A proteomic analysis of the vacuolar content has revealed that chaperones and proteases are over-represented in the PV, suggesting an important role of the vacuole in both protein folding and processing (Nyalwidhe and Lingelbach, 2006). Whether the PEXEL/HT signal acts to mediate the rate of protein folding, and thus influences protein distribution (possibly by recruitment of chaperones) remains to be experimentally investigated.

Since the discovery of the PEXEL/HT sequence, several proteins have been identified that are transported to the host cell, despite the absence of a recognizable PEXEL/HT signal (Spielmann et al., 2006; Dixon et al., 2008). It is still unclear whether these proteins traverse the same trafficking pathway as PEXEL/HT-positive proteins, and thus one further application of our experimental system is to investigate the effect of protein folding on transport of such proteins.

To sum up, our data strongly support the existence of a VPCC within the PVM, which requires proteins to be unfolded before passing into the erythrocyte cytoplasm. The exact molecular identity of the VPCC remains elusive and from our data it is not possible to predict whether the VPCC developed from a prior existing protein translocon that has evolved to cope with a new function (which would seem, in evolutionary terms, the most likely scenario), or represents a totally new class of protein translocons. Bioinformatics have not yet revealed any promising candidates, but this may be due to the genetic distances involved, low conservation at the amino acid level, and the difficulties involved in prediction of both channel and beta-barrel proteins from sequence data alone (S. Charpian and J.M. Przyborski, unpubl. data). While our data do not directly allow the identification of the VPCC, they may provide the necessary tools with which to isolate and characterize this translocon, possibly by the generation of translocation intermediates with the mDHFR system, followed by cross-link and MALDI-TOF analysis. Molecular identification of the VPCC may also provide a promising new target for the development of antiplasmodial compounds, by blocking the transport of essential parasite proteins that are trying to ‘break on through to the other side’.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Vector design/plasmid construction

Primers used in the construction of transfection vectors are listed in Table S1. pARL2-GFP: The coding region of GFPmut2 was PCR amplified from pARL-GFP (Przyborski et al., 2005) using Phusion polymerase (New England Biolabs) and the primers GFP-K-F and GFP-X-R, digested with XmaI and KpnI (NEB) and ligated into XmaI/KpnI-restricted pARL2. pARL2-DG: The coding region of mDHFR was obtained by PCR with primers DHFR-X-A-F and DHFR-K-R using FGF2-GFP-DHFR (Backhaus et al., 2004) as a template. Following KpnI and AvrII digestion, the resulted product was ligated into KpnI/AvrII-restricted pARL2-GFP. pARL2-SDG: Primers stev-X-F and stev1-80-B-A-R were designed to amplify STEVOR1-80 in a PCR reaction using pARL-STEVOR1-80 as a template (Przyborski et al., 2005). The product was digested with XhoI and AvrII and ligated into XhoI/AvrII-restricted pARL2-DG. pARL2-GDG: GBP-Xho-F and GBP150-B-R were used as primers to generate a cDNA sequence encoding the first 150 amino acids of GBP130 from 3D7 total RNA using SuperScript II RT kit (Invitrogen). The resulted product was, after digestion with XhoI and BssHII, ligated into XhoI/BssHII-digested pARL2-SDG. pARL2-GG: pARL2-GDG was digested using XhoI and BssHII to obtain the insert encoding for the first 150 amino acids of GBP130. The product was then ligated into XhoI/BssHII-restricted vector pARL2-GFP. All constructs were verified by automated DNA sequencing and restriction digest analysis.

Parasite cultivation and transfection

Transfection and maintenance of P. falciparum clone 3D7 was carried out as previously described (Trager and Jensen, 1976; Voss et al., 2006). Parasites were synchronized using gelafundin flotation (late-stage parasites) (Pasvol et al., 1978) or sorbitol (ring stages) as previously described (Lambros and Vanderberg, 1979).

Live-cell imaging and indirect immunofluorescence assay (IFA)

Immunofluorescence assay (IFA) experiments were carried out as previously described (Tonkin et al., 2004), except samples were fixed at 37°C for 30 min. Antibodies used: mouse monoclonal anti-GFP (Roche), rabbit anti-PfPV1 (Nyalwidhe and Lingelbach, 2006), monoclonal mouse anti-PfHSP70-1 (a gift of Thierry Blisnick), anti-rabbit Cy3/anti-mouse Cy2 (DAKO). All antibodies were used at 1:1000 dilution. Additionally, parasite nuclei were stained with Hoechst (Invitrogen). For live-cell imaging gelafundin-enriched parasites stained with Hoechst 33258 were directly applied either on a glass slide or to poly-l-lysine-coated glass-bottom culture dishes and imaged immediately at room temperature. Images were acquired using the appropriate filter sets on a Zeiss Axio observer inverse epifluorescence microscope system. For time-lapse experiments, acquisition was carried out at the fastest speed possible, reduced light intensity to decrease bleaching. Signal amplification was set to 16 to increase sensitivity and allow use of lower exposure times. Images used to provide a direct comparison between cells following different treatment were acquired using constant settings. For the live-cell SLO treatment, SLO was added and imaging started immediately following lysis of non-infected red blood cells. For experiments using BFA (Sigma), young trophozoite-stage parasites were treated with BFA at a final concentration of 5 μg ml−1 for 6 h prior to observation.

Image processing and presentation

Individual images were imported into Image J64 (version 1.39u, available at http://rsb.info.nih.gov/ij), converted to 8-bit greyscale, subjected to background subtraction and overlaid. For the experiments presented in Fig. 10, GFP time series were subjected to bleach correction using a macro developed by J. Rietdorf (EMBL Heidelberg, Germany: available at http://www.embl-heidelberg.de/eamnet/html/body_bleach_correction.html). False colouring to increase contrast was carried out using the brgbcmyw look-up table integrated into Image J. To create figures, TIF files were imported into Powerpoint (Microsoft), assembled and slides exported as TIFs. No gamma adjustments were applied to any images, and all data are presented in accordance with the recommendations of Rossner and Yamada (2004).

Protein analysis

Cell fractionation using SLO (kindly provided by Professor S. Bhakdi) was carried out as described previously (Ansorge et al., 1996). Following SLO lysis, the cell pellet was washed six times in PBS, lysed in 10 mM Tris/1 mM EDTA/PIC (Calbiochem) and subjected to three cycles of freezing/thawing. The soluble fraction of the pellet was collected by centrifugation and equal equivalents of the soluble pellet fraction and SLO supernatants (equivalent to 1 × 107 cells) were analysed by SDS-PAGE and immunoblot analysis, using rabbit anti-SERP (1:500) (Ansorge et al., 1996), mouse anti-GFP (1:1000, Roche), rabbit anti-PfGBP130 (Ansorge et al., 1996), mouse anti-PfHSP70 (1:1000, a gift of T. Blisnick), mouse anti-mDHFR (1:200, Abcam) or mouse anti-HsHSP70 (1:1000, Santa Cruz) followed by either HRP-conjugated anti-mouse or anti-rabbit antibodies (DAKO, Santa Cruz, respectively, 1:2000).

Protease protection assays

Transfectant parasites at trophozoite stage expressing the cytosolic fusion protein DHFR–GFP were isolated using saponin lysis. For the assay the soluble parasite fraction of cell equivalents corresponding to 1 × 107 was used. Soluble fractions were subjected to different folate analogues, using, as indicated in the figure, 10 or 100 μM AP (Sigma), 100 μM WR (Jacobus Pharmaceuticals) or equivalent amounts of DMSO (Fluka) as a negative control then incubated on ice for 10 min followed by an incubation period of 14 h at 37°C.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We wish to thank Thierry Blisnick for the gift of anti-PfHSP70 antibodies, Walter Nickel for the mDHFR coding sequence, and Uwe. G. Maier for critical reading of the manuscript. This work was supported by DFG Grant PR1099/1-1 to J.M.P. as part of the SPP1131 priority programme. N.G. is an associate student of the DFG Graduate School GK1216/IITC. I.M. is supported by the Fazit-Stiftung. We are grateful to the co-workers at the Blood Bank of the University Clinic Marburg for their support.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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MMI_6552_sm_Info_and_Table_S1_and_Figures_S1-S2.pdf863KSupporting info item
MMI_6552_sm_Movie_S1.avi2552KSupporting info item

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